The IKK-2/I�B�/NF-�B Pathway Plays a Key Role in the Regulationof CCR3 and eotaxin-1 in FibroblastsA CRITICAL LINK TO DERMATITIS IN I�B�-DEFICIENT MICE*

Received for publication, September 27, 2001, and in revised form, October 25, 2001Published, JBC Papers in Press, November 2, 2001, DOI 10.1074/jbc.M109358200

Margit A. Huber‡§, Andrea Denk§, Ralf U. Peter‡, Lutz Weber‡, Norbert Kraut¶�,and Thomas Wirth§�**

From the ‡Department of Dermatology, Ulm University, Oberer Eselsberg 40, 89081 Ulm, Germany, the §Department ofPhysiological Chemistry, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany, and the ¶Department ofExploratory Research, Boehringer Ingelheim Austria GmbH, Dr. Boehringer-Gasse 5–11, 1121 Vienna, Austria

Tumor necrosis factor (TNF)-�-induced phosphoryla-tion of the I�B proteins by the I�B kinase (IKK) complexcontaining IKK-2 and subsequent degradation of the I�Bproteins are prerequisites for NF-�B activation, result-ing in the stimulation of a variety of pro-inflammatorytarget genes. The C-C chemokine eotaxin-1 is a potentchemoattractant for eosinophils and Th2 lymphocytes,may play an important role in the pathogenesis of atopicdermatitis, and acts via binding to its receptor CCR3. Toinvestigate the role of NF-�B signaling in the regulationof these genes, we stably expressed a transdominantmutant of I�B� and a constitutively active mutant ofIKK-2 in mouse NIH3T3 fibroblasts. The transdominantI�B� mutant completely inhibited TNF-�-mediated in-duction of both eotaxin-1 and CCR3, whereas expressionof constitutively active IKK-2 was sufficient to drivealmost full expression of these two genes in the absenceof TNF-�. Moreover, we observed elevated expressionlevels of CCR3 and eotaxin-1 protein levels in the skin ofI�B�-deficient mice characterized by a widespread der-matitis. Finally, using dermal fibroblasts derived fromI�B�-deficient mice, we observed elevated basal expres-sion, enhanced inducibility by TNF-�, and attenuateddown-regulation upon TNF-� withdrawal of both CCR3and eotaxin-1 mRNA levels. These results demonstratethat the IKK-2/I�B�/NF-�B pathway plays a critical rolefor CCR3 and eotaxin-1 expression in fibroblasts andsuggests a critical link to the pathogenesis of atopicdermatitis.

The NF-�B1 transcription factor family is the most criticalregulator of immediate transcriptional responses in inflamma-tory situations. Rel family members (p65/RelA, RelB, c-Rel,p50, and p52) form homo- or heterodimeric complexes witheach other that constitute the NF-�B complex (1, 2). The crit-ical role of NF-�B family members for distinct cellular func-

tions, such as cell proliferation, cytokine gene expression, orprotection from apoptosis, has been revealed by gene knockoutexperiments (2). In addition, there is increasing evidence for arole of NF-�B transcription factors in different pathophysiolog-ical processes, including atherosclerosis and cancer (3). In rest-ing cells, NF-�B is inactive because of association with inhibi-tor �B (I�B) proteins that mask the nuclear localizationsequence of NF-�B, thereby retaining it in the cytoplasm andpreventing DNA binding. Several I�B proteins are involved inthe control of NF-�B activity, three of these, i.e. I�B�, I�B�,and I�B�, act in a stimulus-dependent manner. Upon inflam-matory activation, I�B is phosphorylated in its N-terminaldomain; subsequently it becomes ubiquitinylated and finallydegraded by the proteasome. This allows nuclear translocationof NF-�B and binding to cognate DNA motifs in the promoterregion of target genes, which subsequently initiates transcrip-tion. The critical step in NF-�B activation is the phosphoryla-tion of I�B by a large multisubunit kinase complex consisting ofI�B kinases (IKK) 1/� and 2/� as well as an additional essentialprotein, NEMO/IKK� (reviewed in Ref. 2). NEMO representsthe regulatory component of the IKK complex, whereas IKK1and IKK2 act as catalytic subunits. Both IKKs can phospho-rylate all three I�B proteins (�, �, and �) to a similar extent;however, from gene knockout experiments it became clear thatIKK2 plays the dominant role in signal-induced phosphoryla-tion/degradation of I�B proteins (reviewed in Ref. 2). I�B deg-radation and subsequently NF-�B activity can be induced inmany cell types by different stimuli. Several parallel signaltransduction pathways appear to exist, all of which ultimatelyresult in IKK activation and I�B degradation (Ref. 4 and ref-erences therein). Among the best understood signaling path-ways are the ones for the inflammatory cytokines tumor necro-sis factor (TNF)-� and interleukin (IL)-1 (5).

Chemokines are a large family of small proteins involved inthe activation and recruitment of specific cell populations dur-ing disease (reviewed in Ref. 6). Eotaxin-1 is a potent eosino-phil chemoattractant belonging to the class of C-C chemokines(7). The protein is potent in inducing eosinophil accumulationin vivo (7, 8). Eotaxin-1 knockout mice demonstrate thateotaxin-1 enhances the magnitude of the early eosinophil re-cruitment after allergen challenge in models of asthma, eventhough the suppression of eosinophil accumulation in chal-lenged/sensitized mice was only partial (reviewed in Ref. 9).Eotaxin-1 expression was found to be restricted to a few celltypes, including eosinophils, bronchial epithelial cells, and der-mal fibroblasts (reviewed in Refs. 9 and 10). Its expression hasbeen found to be enhanced in these cell types in asthmatics,and increased expression is associated with disease severity

* This work was supported by Grants Wi789/2 and Wi789/3 from theDeutsche Forschungsgemeinschaft and by the Fonds der ChemischenIndustrie (to T. W.). The costs of publication of this article were de-frayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

� These authors contributed equally to this work.** To whom correspondence should be addressed. Tel.: 49-731-500-

23270; Fax: 49-731-500-22892; E-mail: thomas.wirth@medizin.uni-ulm.de.

1 The abbreviations used are: NF-�B, nuclear factor-�B; AD, atopicdermatitis; CA, constitutively active; I�B, inhibitor �B; IKK, I�B ki-nase; IL, interleukin; RT, reverse transcriptase; TD, transdominant;TNF-�, tumor necrosis factor-�; PBS, phosphate-buffered saline.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 2, Issue of January 11, pp. 1268–1275, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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(11, 12). Moreover, eotaxin-1 expression in epithelial cells wasfound to be increased in atopic dermatitis (13), as well as inother inflammatory conditions (14). The expression of eotaxin-1can be induced by TNF-� in epithelial cell lines, such as A549cells (15), and by IL-1� and TNF-� in fibroblasts (16, 17). Incontrast to most other eosinophil chemoattractants of the CC-chemokine family that generally act on several receptors,eotaxin-1 only signals via one specific chemokine receptor,namely the G protein-coupled receptor CCR3 (reviewed in Ref.18). CCR3 is prominently expressed on eosinophils, basophils,Th2-type lymphocytes, and fibroblasts (reviewed in Refs. 10and 12). Based on these findings, an analysis of the regulationof CCR3 and eotaxin-1 expression in fibroblasts, in particularin dermal fibroblasts, is likely to yield information relevant tothe pathogenesis of allergic inflammation such as atopicdermatitis.

Although very little is known on how CCR3 is regulated atthe transcriptional level, there is increasing evidence thatNF-�B may be involved in the regulation of eotaxin-1 expres-sion. Firstly, NF-�B elements are present in the eotaxin-1promoter in both humans and mice (17, 19). Second, mouseknockouts lacking the p50 subunit of NF-�B show no eotaxin-1induction in response to ovalbumin challenge (20). Third,eotaxin-1 promoter activity was increased by TNF-�, and anNF-�B binding site was shown to be critical for this inductionin the airway epithelial cell line BEAS-2B (21). Finally, anNF-�B binding site in the eotaxin-1 promoter was shown to becritical for the induction of eotaxin-1 by IL-1� in A549 airwayepithelial cells (22). Even though these observations point to-ward an important role of NF-�B in eotaxin-1 regulation, nei-ther of these reports has demonstrated whether or not thissignaling pathway is essential in a stimulus-dependent man-ner and/or in an in vivo situation.

Here, we have investigated the role of NF-�B signaling forthe regulation of CCR3 and eotaxin-1, two key regulators ofatopic inflammatory responses. Upon exogenous expression ofCA IKK-2 and TD I�B� in the fibroblast cell line NIH3T3, weshow that NF-�B signaling is critical for the induction of CCR3and eotaxin-1 in response to TNF-�. Using I�B�-deficient mice,we provide evidence that CCR3 and eotaxin-1 are physiologicaltargets of NF-�B signaling in vivo and for their up-regulationin I�B�-deficient skin that may contribute to the skin pathol-ogy resembling atopic dermatitis.

EXPERIMENTAL PROCEDURES

Cells and Cell Culture—NIH3T3 (a kind gift from Dr. Garin-Chesa,Boehringer Ingelheim Pharma KG), mouse embryonic fibroblasts,mouse dermal fibroblasts (isolation described below), and �NX ampho-tropic retrovirus producer cells (a kind gift from G. Nolan, Stanford, CA)were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) con-taining 10% fetal calf serum (PAN Systems, Aidenbach, Germany), 100units/ml penicillin, and 100 �g/ml streptomycin (Invitrogen) at 37 °C,5% CO2. For stimulation experiments human recombinant TNF-� (agift from Dr. Adolf, Boehringer Ingelheim) was dissolved in a buffercontaining 10 mM sodium phosphate, pH 7, 200 mM sodium chloride,and 2 mg/ml bovine serum albumin and used at the indicated concen-trations. At least 12 h prior to stimulation, the cells were held instarvation medium consisting of Dulbecco’s modified Eagle’s mediumcontaining 0,5% fetal calf serum, 100 units/ml penicillin, and 100 �g/mlstreptomycin.

Stable Transfection of NIH3T3 with 3x�B Luciferase Reporter andLuciferase Activity Assay—For generation of stable transfectants of theNIH3T3 cell line, cells were electroporated (Bio-Rad gene pulser) with20 �g of the 3x�B.luc reporter plasmid together with 1 �g of a pSV.purovector (conferring resistance to puromycin) at 250 microfarad and 450V. After electroporation, the cells were immediately resuspended inmedium and seeded in 10-cm tissue culture plates. Cell clones with anintegrated reporter gene were selected in medium containing 6 �g/mlpuromycin with selection starting 48 h after electoporation. After 10–14days single clones were picked and expanded. For measurement ofluciferase activity cells were harvested, and luciferase activity was

determined using the Lumat LB 9507 (Berthold Technologies, BadWildbad, Germany).

Retroviral Vectors and Stable Producer Cell Lines—The pCFG5 IEGZretroviral vector used for infection has been described earlier (23). AllcDNAs were inserted into blunted EcoRI/BamHI sites. Mutant I�B�was provided by Patrick Baeuerle (Micromed, Munich, Germany) andthe mutant IKK-2 cDNA by Alain Israel (Institut Pasteur, Paris,France). Sequences of retroviral vectors were confirmed by DNA se-quencing. �NX producer cells plated at a density of 1 � 106/10-cm platewere transfected using the calcium phosphate precipitation methodwith 10 �g of plasmid DNA as described (24). 24 h later, transfectionefficiencies were determined by monitoring green fluorescent proteinexpression by fluorescence microscopy (Improvision, Heidelberg, Ger-many). Transfection efficiencies usually ranged between 70 and 80%.24 h after transfection, 1 mg/ml zeocin (Invitrogen) was added to thecells, which were then grown in the presence of this agent for another2 weeks until all the cells were positive for green fluorescent protein.

Retroviral Infection of NIH3T3 3x�B with Supernatant from �NXProducer Cells—One day before infection, NIH3T3 cells were seeded insix-well plates at a density of 2 � 105 cells/well, and the �NX cells wereseeded at a density of 3 � 106/10-cm plate. At the day of infection, �NXcell supernatant was obtained and filtered through a 0.45-�m filter, and5 �g/ml polybrene (Sigma) was added to the filtrate. Thereafter, me-dium was removed from NIH3T3 cells and replaced by �NX cell super-natant containing the retrovirus. Culture plates were centrifuged at1000 � g for 3 h, and supernatants then removed and replaced byconventional Dulbecco’s modified Eagle’s medium. 48 h later the effi-ciency of infection was monitored by fluorescence microscopy as de-scribed above (infection efficiencies of NIH3T3 cells ranged between 80and 90% depending on the retrovirus used), and selection with zeocin(1000 �g/ml) was started.

Western Blot Analysis and Electrophoretic Mobility Shift Assay—Preparation of whole cell extracts was performed as described earlier(25). For Western blot analysis, 50 �g of protein extracts/lane wereseparated on 12.5% polyacrylamide gels and transferred onto polyvi-nylene difluoride membranes (Millipore, Bedford, MA). The membraneswere blocked with 7.5% dry milk in PBS containing 0.2% Tween 20. Forsubsequent washes, 0.2% Tween 20 in PBS was used. The membraneswere labeled with affinity-purified rabbit antiserum against I�B� orIKK-2 (Santa Cruz Biotechnology, Santa Cruz, CA). Thereafter, themembranes were stained with horseradish peroxidase-coupled second-ary donkey anti-rabbit IgG antibody (Dianova, Hamburg, Germany)that was visualized by enhanced chemiluminescence (ECL; AmershamBiosciences, Inc.). As a loading control, the membrane was incubatedwith stripping buffer (40 min, 56 °C) and, after extensive washing withPBS containing 0.2% Tween 20, labeled with rabbit polyclonal antibodyagainst p65 (Santa Cruz Biotechnology). After incubation with second-ary donkey anti-rabbit IgG antibody and washing with PBS containing0.2% Tween 20, chemiluminscent substrate was added, and the mem-brane was subjected to autoradiography (as described above). Electro-phoretic mobility shift assays were performed essentially as describedbefore (25).

Semiquantitative RT-PCR—Total RNA was extracted, and semi-quantitative RT-PCR was carried out as described earlier (10). MouseCCR3 was amplified with primers 5�-CAA CTT GGC AAT TTC TGACCT G-3� (sense) and 5�-GCA AAC ACA GCA TGG ACG ATA G-3�(antisense; 37 cycles); mouse eotaxin-1 was amplified with primers5�-CAA CAG ATG CAC CCT GAA AGC-3� (sense) and 5�-TCC CTG AGAGCA CGT CTT AGG A-3� (antisense; 37 cycles); mouse EF-1� wasamplified with primers 5�-AGT TTG AGA AGG AGG CTG CT-3� (sense)and 5�-CAA CAA TCA GGA CAG CAC AGT C-3� (antisense; 23 cycles);all primers were obtained from MWG Biotech (Ebersberg, Germany).I�B�-specific primers were a kind gift from A. Beg (Columbia Univer-sity, New York, NY) and were used for genotyping of I�B� knockoutmice.

I��� Knockout Mice and Isolation of Dermal Fibroblasts—Mice witha genetic deletion of I�B� have been described (26, 27). The mice usedhere were a kind gift from Amer Beg (Columbia University). GenomicDNA was prepared from tails of 7-day-old pups and analyzed usingI�B�-specific primers. Samples from the skin were taken and digestedwith trypsin (Invitrogen) for 30 min with occasional mixing. Afterward,the samples were further homogenized by pushing them through asyringe. Then the cells were seeded in a 10-cm tissue culture plate. Thecells were grown for several weeks until they went through a crisis andwere spontaneously transformed.

Immunohistochemistry—Biopsy specimens of mouse skin were em-bedded in OCT compound (Tissue-Tek, Miles Inc., Elkhart, state),quick-frozen in liquid nitrogen, and stored at �80 °C. Cryostat sections

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(5 �m) were cut, mounted on gelatin-coated slides, and fixed in coldacetone (4 °C) for 10 min. The avidin-biotin complex immunoperoxidaseprocedure was carried out as described before (28). The slides wereincubated with goat polyclonal antibody against Eotaxin-1 and goatpolyclonal antibody against CCR3 (both obtained from Santa CruzBiotechnology). The sections were counterstained with Harris’hematoxylin.

RESULTS

Retroviral Transduction of NIH3T3 Cells with DominantInterfering Mutants of the IKK/I�B� Pathway—Some earlierreports had suggested a role of NF-�B in the regulation ofeotaxin-1 expression in airway epithelial cells (21). We wantedto determine whether eotaxin-1 and CCR3 are inducibly ex-pressed in fibroblasts and what role, if any, NF-�B signalingplays in such a scenario. To analyze CCR3 and eotaxin-1 in-ducibility, we chose primary mouse embryo fibroblasts thatwere treated for 4 or 24 h with TNF-�. RT-PCR analysis re-vealed that the expression of both genes was up-regulated uponstimulation with TNF-� and that stimulation for 24 h resultedin no further induction compared with stimulation for 4 h (Fig.1A). Similarly, CCR3 and eotaxin-1 were up-regulated in themouse fibroblast cell line NIH3T3 upon addition of TNF-�.Although stimulation with TNF-� for 1 h resulted in only aweak induction of these two genes, a 4-h stimulation periodresulted in high expression levels of CCR3 and eotaxin-1 (Fig.1B). These results provided a first hint that NF-�B signalingmay be involved in the regulation of these two genes infibroblasts.

To correlate all subsequent modulations of the NF-�B sig-naling pathway directly to NF-�B function, we engineeredNIH3T3 fibroblasts that stably express a luciferase gene underthe control of multimerized �B-sites (Fig. 2A). Treatment ofindividual stably transfected clones with TNF-� resulted in astrong up-regulation of luciferase activity, and the best clone(clone 12) showed an almost 250-fold induction upon addition ofTNF-� (Fig. 2B). This clone was then infected with retrovirusesexpressing either a TD I�B� protein (serines 32 and 36 aremutated to alanine), a constitutively active (CA) IKK-2 protein(two serines in the activation loop are mutated to glutamic acidresidues), or an empty vector control (Fig. 2C). Stably infectedcells could be visualized by immunofluorescence microscopy asthe retroviruses coexpress enhanced green fluorescent protein.As shown in Fig. 2D, the infection rate of NIH3T3 clone 12 cells

with these three constructs was close to 90% even prior toselection. Expression of TD-I�B� and CA-IKK-2 was controlledby Western immunoblotting (Fig. 2E). This analysis revealedstrong overexpression of the mutant proteins as compared withthe endogenous counterparts. In the presence of high levels ofexogenous TD-I�B�, expression of endogenous I�B� was barelydetectable. This is most likely due to reduction in NF-�B activ-ity, resulting in a decrease in I�B� synthesis. High levels ofCA-IKK2 also resulted in decreased levels of endogenous I�B�.This can be explained by constitutive stimulation of I�B� phos-phorylation, resulting in its degradation. Moreover, overex-pression of CA-IKK-2, but not of TD-I�B�, resulted in slightlyelevated levels of endogenous IKK-1 (Fig. 2E).

Dominant Interfering Mutants in IKK-2 and I�B� Show ThatActivation of NF-�B Is Critical for Inducible CCR3 andeotaxin-1 Expression in NIH3T3 Fibroblasts—The conse-quences of TD-I�B� and CA-IKK-2 expression on NF-�B activ-ity were analyzed by luciferase assays and electrophoretic mo-bility shift assay. Although NIH3T3 clone 12 cells stablyinfected with an empty vector showed a more than 100-foldinduction of luciferase activity upon stimulation with TNF-�,cells infected with TD-I�B� displayed no significant luciferaseactivity, regardless of whether TNF-� was present or absent(Fig. 3A). In contrast, cells infected with CA-IKK-2 exhibited ahigh level of luciferase activity already in the absence of TNF-�(25-fold higher than empty vector-infected cells), which couldnot be further elevated upon addition of TNF-�. Similar resultswere obtained by monitoring DNA binding activity of NF-�B inthese cells (Fig. 3B). In the presence of TD-I�B�, no detectableNF-�B DNA binding activity could be induced by TNF-�. Incontrast, the CA-IKK-2 expressing cells showed already con-siderable NF-�B DNA binding activity in the absence of TNF-�,which was, however, further increased at the 4-h time point.We then asked whether this NF-�B modulation affected theinducible expression of CCR3 and eotaxin-1 in NIH3T3 fibro-blasts. RT-PCR analyses revealed that infection with the con-trol vector did not affect the expression/induction of the tran-scripts of these two genes (Fig. 3C). In contrast, in cells infectedwith TD-I�B� protein, expression could not be induced bytreatment of the cells with TNF-�. Importantly, cells express-ing the CA-IKK-2 protein showed a high expression level ofthese two genes in the absence of TNF-�. After 4 h of TNF-�treatment, the inducible expression level was slightly in-creased further. These results demonstrate that activation ofNF-�B is critical for the induction of CCR3 and eotaxin-1 geneexpression in fibroblasts and that selective activation of thispathway already partially activates expression of these genes.

Elevated Basal Expression, Enhanced Inducibility, and At-tenuated Down-regulation of CCR3 and eotaxin-1 in Fibro-blasts Isolated from I�B�-deficient Mice—Given the criticalrole that NF-�B plays for CCR3 and eotaxin-1 expression, weasked whether expression of these genes is affected in dermalfibroblasts of mice lacking the I�B� protein. Mice bearing ahomozygous mutation in the gene coding for I�B� die about7–8 days after birth because of a massive myeloproliferativedisorder (26). Fig. 4A shows the genotyping of 7-day-old I�B�

wild-type, heterozygous, and homozygous mutant mice (ob-tained by A. Beg, Columbia University). As already described(26, 27), I�B�-homozygous null pups at day 7 are significantlysmaller than their littermates and show a widespread derma-titis characterized by xerosis, scaling plaques, and lichenifica-tion (Fig. 4, B and C). Next we analyzed skin sections (stainedwith hematoxylin-eosin) of 7-day-old wild-type (�/�), heterozy-gous (�/�), and homozygous null (�/�) animals. The wild-typeskin (Fig. 5A) displays the normal aspect of a 7-day-old murineskin with an intact epidermis comprising morphologically nor-

FIG. 1. Induction of CCR3 and eotaxin-1 by TNF-� in fibro-blasts. Primary mouse embryo fibroblasts (A) and NIH3T3 fibroblasts(B) were stimulated with 40 ng/ml TNF-� for the time intervals indi-cated, and RT-PCR analysis was carried out as described under “Ex-perimental Procedures.”

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mal keratinocytes, a pronounced granular zone, and an ortho-keratotic basket-woven stratum corneum. The dermis is welldemarcated and reveals a texture of loosely woven collagenfibers and an intense infiltrate of numerous fibroblasts andhistiocytes, as well as some lymphocytes and plasma cells, yet

hardly any granulocytes. The zone of the subcutaneous fattissue as well as the underlying muscle fibers are morphologi-cally normal. The dermis and subcutaneous fat tissue presentwith a high amount of normally differentiated hair follicles andsebaceous gland units. Although skin from 7-day-old �/� pups

FIG. 2. Retroviral transduction ofNIH3T3 fibroblasts with dominantinterfering mutants of the IKK/I�B�/NF-�B pathway. A, schematic represen-tation of the 3x�B.luc reporter stablytransfected into NIH3T3 fibroblasts. B,upon stable transfection, individualclones were assayed for their luciferaseactivity in the absence or upon stimula-tion with 40 ng/ml TNF-� for 4 h (leftpanel). R.L.U., relative light units. Thefold induction upon TNF �-stimulation isindicated in the right panel. Among theclones showing the best response, clone 12showed the highest inducibility (246�)upon TNF-� addition and was selected forfurther studies. C, schematic representa-tion of the retrovirus used for the expres-sion of TD I�B� or CA IKK-2 mutants(Modulators). IRES, internal ribosomeentry site; LTR, long terminal repeat;Zeo, zeocin. As a control, a retroviruswithout a modulator insert was used.NIH3T3 clone 12 cells were infected withparental vector or retroviruses expressingthe dominant interfering mutants as de-scribed under “Experimental Proce-dures.” D, 48 h after infection, stablyinfected cells were visualized by immuno-fluorescence microscopy for enhancedgreen fluorescent protein expression. E,to determine the expression levels of dom-inant interfering mutants compared withtheir endogenously expressed wild-typecounterparts, infected NIH3T3 clone 12cells were stimulated with TNF-� (40 ng/ml) for 2 h, and whole cell lysates wereprepared for Western blot analysis, usingan IKK- and I�B�-specific antibody si-multaneously for visualization. Proteinbands of CA-IKK-2, endogenous IKK-1,TD-I�B�, and endogenous I�B� are indi-cated. The blot was subsequently strippedand reprobed with a p65/RelA antibody,to monitor equal loading (lower panel).

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(Fig. 5B) shows a variable slight increase in infiltrating leuko-cytes (that has not been described previously), no major differ-ence in terms of the epidermal, dermal, and subcutaneous

architecture was noted. In contrast, a skin cross-section froman I�B�-deficient pup (Fig. 5C) differs notably from both thewild-type and heterozygous skin. Overall, there is a strikingdegree of architectural disorganization in particular with re-gard to the dermal and subcutaneous fat layer; the epidermalgranular zone is less pronounced and the pattern of cornifica-tion is more stratified than basket-woven. The cutaneous/sub-cutaneous border is hardly discernible, and the subcutaneousfat tissue appears remarkably reduced, whereas the musclebundles are well developed. The dermal connective tissue isinterspersed by a dense infiltrate comprising fibroblasts, lym-

FIG. 3. Activation of NF-�B is critical for CCR3 and eotaxin-1 expression in NIH3T3 fibroblasts. A, NIH3T3 clone 12 cells stablytransfected with 3x�B.luc and subsequently stably infected with either the empty vector control, TD-I�B�, or CA-IKK2 were stimulated with 40ng/ml TNF-� for 8 h, and luciferase activity was determined. Luciferase activity is calculated from three independent measurements and indicatedas the mean in relative light units (R.L.U.). The fold induction in the presence of TNF-� is indicated above the bars indicating activity. B, wholecell lysates (6 �g) from NIH3T3 clone 12 cells stably infected with the indicated constructs and treated as indicated were incubated with a�B-specific probe. The positions of the induced NF-�B complexes are indicated. The lower band indicates a nonspecific binding complex and canbe used as an internal loading control. All lanes were loaded equally, except the far right lane (IKK-2, 4 h, control), which was underloaded,resulting in an underrepresentation of the NF-�B binding activity. C, time dependence of CCR3 and eotaxin-1 transcript accumulation in NIH3T3clone 12 cells stably infected with empty vector control, TD-I�B�, or CA-IKK2. Total RNA was extracted from cells either unstimulated (�) orstimulated with 40 ng/ml TNF-� for 1 or 4 h. 100 ng of total RNA was subjected to RT-PCR analysis using gene-specific primers as described under“Experimental Procedures.” Lane 10 shows a control with conditions as in lane 9 lacking cDNA because of omission of reverse transcriptase. CCR3-,eotaxin-1, and EF-1�-specific amplification products are indicated.

FIG. 4. Phenotype of mice bearing a homozygous mutation inthe gene coding for I�B�. A, PCR analysis of tail DNA preparationsfrom offsprings of heterozygous matings. The three genotypic categoriesare indicated as wild-type (�/�), heterozygous (�/�), and homozygous(�/�). The targeted locus results in a 180-bp fragment. B and C,phenotype of I�B� �/� animals. I�B�-homogygous null pups at day 7are characterized by a widespread dermatitis with marked scaling(arrows, B and C) and thickened skin with increased markings, referredto as “lichenification” (indicated by dotted oval in C).

FIG. 5. Histological analysis of skin sections from wild-type,heterozygous, and homozygous I�B�-null pups. Tissues from7-day-old pups were embedded in paraffin and stained with hematox-ylin-eosin for histological analysis. Skin cross-sections from wild-type(A), heterozygous (B), and homozygous mutant pups (C) were evalu-ated. sc, stratum corneum; gz, granular zone; sb, stratum basale; d,dermis; h, hair follicle; ft, fat tissue; ma, microabscess; m, muscle. Seetext for details. Scale bar, 25 �m.

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phocytes, granulocytes, and histiocytes. There is no epidermalspongiosis; however, there are several conspicuous intraepider-mal neutrophilic microabscesses as well as a marked acantho-sis and hyperkeratosis, that are in full accordance with theobservations made by Beg et al. (26) and Klement et al. (27).

We then prepared primary dermal fibroblasts from 7-day-oldwild-type and mutant animals and analyzed expression ofCCR3 and eotaxin-1 after TNF-� stimulation. This analysisrevealed several interesting results (Fig. 6A). First, the basalexpression of both genes was higher in mutant fibroblasts ascompared with wild type. A similar elevation of other cytokinesand chemokines including granulocyte-colony stimulating fac-tor, murine macrophage inflammatory protein-2, and TNF-� inI�B�-deficient pups has previously been described (26, 27).Second, the kinetics of the induction was altered, and bothgenes showed already full level expression after 1 h, whereasthis full expression was only seen after 4 h in control fibro-blasts. Finally, when cells were induced with TNF-� for 4 h,and TNF-� was then removed, CCR3 and eotaxin-1 expressionwas rapidly down-regulated in wild-type fibroblasts. Thisdown-regulation was attenuated in the I�B�-deficient dermalfibroblasts. The attenuated down-regulation of CCR3 andeotaxin-1 in I�B�-deficient fibroblasts is consistent with a de-layed down-regulation of NF-�B DNA binding activity as evi-dent from electrophoretic mobility shift assay experiments(Refs. 26 and 27 and data not shown).

Elevated CCR3 and Eotaxin-1 Protein Levels in Skin of I�B�-deficient Mice—We saw a low but consistent basal expression ofCCR3 and eotaxin-1 in unstimulated I�B�-deficient fibroblasts.Therefore we asked whether an increased expression of thesegenes was detectable in the skin of the mutant animals byimmunohistochemistry. This analysis demonstrated an in-creased staining for antibodies to CCR3 in the skin from I�B�

�/� pups, particularly in suprabasal layers but also in dermalfibroblasts (Fig. 6, B and C). Eotaxin-1 protein expression wasalso significantly elevated in I�B� �/� pup skin (Fig. 6, D andE) and offers a possible explanation for the enhanced infiltra-tion of leukocytes in the dermis of these pups. In summary,these results demonstrate that NF-�B signaling via IKK-2 andI�B� is critical for the basal expression and TNF-�-dependentinduction of CCR3 and eotaxin-1 in fibroblasts.

DISCUSSION

The activation of eotaxin-1 and its receptor CCR3 is thoughtto be a critical step in the onset of inflammatory reactionsassociated with allergic asthma or atopic dermatitis. Here wehave analyzed the contribution of the IKK-2/I�B�/NF-�B sig-naling pathway to CCR3 and eotaxin-1 expression upon inflam-matory stimulation in fibroblasts, a cell type central to inflam-matory conditions. We used a retroviral transduction approachthat allowed the expression of dominant interfering mutants ofcomponents of the NF-�B signaling pathway. The conse-quences of this modulation of NF-�B activity on the expressionof endogenous CCR3 and eotaxin-1 in NIH3T3 fibroblasts wasanalyzed. Using this approach, we were able to demonstratethat the inhibition of NF-�B signaling by expression of a trans-dominant mutant of I�B� results in an almost complete block-ade of TNF-�-induced expression of CCR3 and eotaxin-1. Incontrast, a constitutively active version of IKK-2 by itself wassufficient to induce maximal expression of these genes in theabsence of TNF-�. Interestingly, I�B� mutant skin cells, in-cluding fibroblasts, exhibited elevated levels of CCR3 andeotaxin-1 protein levels, and also the analysis of dermal fibro-blasts derived ex vivo from these I�B�-deficient pups demon-strated a critical role of NF-�B signaling in the regulation ofthese two genes.

FIG. 6. Regulation of CCR3 and eotaxin-1 expression by NF-�B signaling demonstrated by analysis of I�B� mutants. A, primarydermal fibroblasts from I�B� wild-type (�/�) and mutant (�/�) animals were isolated and expanded. The cells were treated with 40 ng/ml TNF-�for the indicated time periods. After 4 h, TNF-� was washed away thoroughly, medium without TNF-� was added, and incubation was continuedfor the indicated time periods. Total RNA was extracted and subjected to RT-PCR analysis using gene-specific primers as described under“Experimental Procedures.” B–E, immunohistochemistry of cryostat skin cross-sections of 7-day-old wild-type (B and D) and homozygous I�B�mutant (C and E) pups. CCR3 and Eotaxin-1 protein levels were determined by incubation with polyclonal antibodies against CCR3 and Eotaxin-1and by performing a routine avidin-biotin-immunoperoxidase staining procedure. Note increased staining of CCR3 in I�B� �/� skin, in particularin suprabasal layers of the epidermis (arrow, C) but also in dermal fibroblasts (arrow, C), whereas only one layer of basal keratinocytes expressesCCR3 in the skin cross-section from a control (B). D and E, increased expression of Eotaxin-1 on keratinocytes and dermal fibroblasts fromI�B�-deficient mice (�/�, arrows, E). Scale bar, 25 �m.

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NF-�B has been previously implicated in allergic inflamma-tion. The majority of proteins encoded by NF-�B target genesparticipate in the host immune responses. These include alarge number of cytokines and chemokines, as well as receptorsrequired for leukocyte adhesion and migration (29). There hasbeen suggestive evidence that NF-�B regulates the chemokineeotaxin-1, a central mediator in recruiting of eosinophils inallergic inflammation. Mochizuki et al. (30) reported thatTNF-�, an inducer of NF-�B signaling, stimulated eotaxin-1expression in fibroblasts. We have found a similar effect ofTNF-� in the induction of eotaxin-1 mRNA in NIH3T3 cells,mouse embryonic fibroblasts, and mouse dermal fibroblasts.Yang et al. (20) have shown that mice deficient in the p50subunit of NF-�B protein do not mount eosinophilic lung in-flammation and that eotaxin-1 expression was inhibited com-pared with that in wild-type mice. These data are in agreementwith our findings indicating an important role for NF-�B in theregulation of eotaxin-1. Furthermore, in vitro studies usingreporter constructs have suggested that overlapping elementsfor NF-�B and Stat6 within the eotaxin-1 promoter mediate thetranscriptional induction by TNF-� and IL-4, respectively, inairway epithelial cells (21). Our data confirm and extend thesefindings and demonstrate in vivo roles for IKK-2, I�B�, andNF-�B in the TNF-�-induced regulation of eotaxin-1. In con-trast to eotaxin-1, very little has been reported on the mecha-nisms of CCR3 regulation so far. Analysis of the CCR3 generevealed a complex 5� exon organization and a broadly activepromoter with eosinophil-selective elements. The CCR3 pro-moter also appears to contain putative NF-�B binding sites,which, however, have not yet been further analyzed (31). Al-though further studies are required to demonstrate whetherCCR3 is a direct target gene of NF-�B, our results show afunctional requirement of IKK-2/I�B�/NF-�B signaling in theregulation of CCR3 expression. To our knowledge, this is thefirst demonstration that NF-�B signaling is critical for TNF-�-mediated induction of CCR3 expression in fibroblasts and pro-vides additional evidence for a role of NF-�B in allergicinflammation.

I�B� is the major ubiquitous cytoplasmic inhibitor that iscritical for regulating the rapid transient nuclear induction ofNF-�B. Although the embryonic development of mice lackingI�B� appears to be normal, I�B� �/� mice die 7–10 dayspostnatally, afflicted by severe widespread inflammatory der-matitis and granulocytosis (26, 27). Coincident with this phe-notype, the expression of certain proinflammatory cytokinesand factors associated with granulocyte recruitment, adher-ence, and activation such as TNF-�, granulocyte-colony stimu-lating factor, murine macrophage inflammatory protein-2, andthe adhesion molecule vascular cell adhesion molecule-1 isincreased. However, not all genes known to be induced byNF-�B are up-regulated in I�B� �/� cells. Our results clearlyprovide evidence that regulation of CCR3 and eotaxin-1 byNF-�B occurs at least in part via repression by I�B�, becauseexpression levels of these two target genes are elevated in theskin of I�B� �/� mice. Furthermore, dermal fibroblasts iso-lated from these mice show elevated basal expression, en-hanced inducibility, and attenuated down-regulation of CCR3and eotaxin-1 expression. Interestingly, despite the absence ofI�B� in these knockout mice, changes in the constitutive nu-clear levels of NF-�B are cell type-dependent. For example,whereas an increase in constitutively nuclear p50/relA and p50homodimers was observed in I�B� �/� thymocytes and spleno-cytes, the levels of constitutive NF-�B complexes were un-changed in I�B� �/� embryonic fibroblasts (26). Our observa-tion of an elevated basal level of the two NF-�B target genes indermal fibroblasts of I�B� �/� mice argues that I�B� plays a

critical role in regulating the cytoplasmic retention of NF-�B inunstimulated dermal fibroblasts. This has not been observed inembryonic fibroblasts derived from these mice (27). Further-more, we detected a prolonged post-induction repression ofCCR3 and eotaxin-1 in dermal fibroblasts derived from I�B�

�/� mice upon removal of TNF-�. Because attenuated down-regulation of NF-�B signaling has also been observed in mouseembryonic fibroblasts of I�B� �/� mice (27), the requirementfor I�B� in termination of the NF-�B response appears to be amore general mechanism. Recent data indicate that activationof IKK-2, rather than IKK-1, participates in the primary path-way by which proinflammatory stimuli induce NF-�B function.IKK-2 has been shown to play a central role in IL-1- andTNF-�-mediated NF-�B activation and expression of proin-flammatory genes in several cell types (reviewed in Ref. 32).Our results indicate that IKK-2 is also a critical regulator ofproinflammatory gene expression in fibroblasts. Activation ofNF-�B leads to the induction of multiple genes, encoding atleast 27 different cytokines and chemokines, receptors involvedin immune recognition, proteins involved in antigen presenta-tion, and receptors required for leukocyte adhesion and migra-tion (reviewed in Ref. 33). Thus, NF-�B activation is assumedto lie at the heart of many inflammatory diseases, such asrheumatoid arthritis, asthma (20), and inflammatory boweldisease (reviewed in Ref. 32). In addition, NF-�B regulationmay be involved in the pathogenesis of diseases such as ather-osclerosis and Alzheimer’s disease, in which the inflammatoryresponse is at least partially involved (reviewed in Ref. 33).Several lines of evidence suggest that NF-�B activation ofcytokine genes is an important contributor to the pathogenesisof atopic asthma, which is characterized by the infiltration ofeosinophils and lymphocytes into the sites of inflammation(34). Many recent in vivo and in vitro studies have implicatedeotaxin-1 in this process (7, 35, 36). Recently it has been dem-onstrated that eotaxin-1 and CCR3 protein expression is sig-nificantly enhanced in lesional skin of patients suffering fromatopic dermatitis (AD) (13). Eotaxin-1 is a potent chemoattrac-tant and activator not only of eosinophils and basophils butalso for Th2 lymphocytes (37), which are associated with theinitial phase of inflammation in AD (38). The suggestion thatNF-�B dysregulation may be a critical factor in mediatingsusceptibility to AD is supported by the findings that RelB-deficient mice show a phenotype and histopathological changesresembling AD (39), accompanied by increased mRNA levels ofeotaxin-1 and CCR3 in lesional skin. It should be noted, how-ever, that the basis of the inflammatory pathology in relB �/�mice may be due to the absence of certain thymic and splenicdendritic cell populations that account for the inability to de-lete autoreactive T cells (40). These T cells may, in a feedbackcycle, stimulate resident cells, e.g. fibroblasts, to release che-mokines and therefore increase leukocyte accumulation intothe affected tissue (39).

Our results suggest a critical role of NF-�B signaling in thepathogenesis of AD for the following reasons. First, the regu-lation of eotaxin-1 and CCR3 by the IKK-2/I�B�/NF-�B path-way is important at the mRNA level in fibroblasts. Second,I�B�-deficient mice are afflicted by a severe widespread der-matitis (Refs. 26 and 27 and our results) that revealed severalhistopathological parallels to AD in humans. Coincident withthis phenotype, the expression of CCR3 and eotaxin-1 in der-mal fibroblasts isolated from lesional I�B �/� skin was en-hanced and prolonged in response to the activation signalTNF-�, and moreover, Eotaxin-1 and CCR3 protein expressionin lesional skin from these animals was markedly induced. Itremains to be elucidated whether fibroblasts in patients withAD show altered p50-relA/NF-�B activity. However, the ele-

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vated expression levels of Eotaxin-1 and CCR3 in I�B�-defi-cient mice offers a likely explanation for the presence of infil-trating leukocytes in the skin of these pups and their skinpathology resembling AD. A detailed characterization of NF-�Bsignaling in AD will be essential to develop specific therapeuticstrategies for atopic diseases such as asthma and AD.

Acknowledgments—We thank Dr. Bernd Baumann for helpful dis-cussions and critical reading of the manuscript, Dragan Marinkovic andTatjana Samardzic for assistence with immunofluorescence, Dr. AmerBeg for the I�B� mutant mice, and U. Leschik, C. Pantic, and E.Peschke for excellent technical assistance.

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WirthMargit A. Huber, Andrea Denk, Ralf U. Peter, Lutz Weber, Norbert Kraut and Thomas

-DEFICIENT MICEαBκ in Fibroblasts: A CRITICAL LINK TO DERMATITIS IN Ieotaxin-1 and CCR3B Pathway Plays a Key Role in the Regulation of κ/NF-αBκThe IKK-2/I

doi: 10.1074/jbc.M109358200 originally published online November 2, 20012002, 277:1268-1275.J. Biol. Chem. 

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